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Chromatin is a remarkably dynamic material, as chromosomal structures formed to silence transcription are remodeled and modified to enable transcription in response to cellular signals. Chromatin transitions are mediated by a set of multi-protein chromatin remodeling complexes, which affect the function and positioning of nucleosomes, the basic repeating unit of chromatin structure. Transcriptional regulation by nucleosomes is potentiated by the acetylation and methylation of lysine residues on the nucleosome 'tails' according to a complex 'histone code'. Chromatin remodeling/modifying complexes include: 1) histone acetyltransferases (HATs), 2) histone methyltransferase (HMTs), and 3) Remodelers. HAT and HMT complexes covalently mark nucleosomes, whereas Remodelers recognize these marks, and then use the energy of ATP hydrolysis to reposition nucleosomes on the DNA, thereby revealing the underlying sequence to transcriptional regulators. We are interested in how Remodelers reposition nucleosomes, how they recognize proper nucleosomes via the histone code. As these complexes are well conserved in all eukaryotes, most our studies are focused in yeast, where potent genetic and genomic tools can be utilized. We are also very interested in understanding how the histone methylation code is established and removed, and have characterized the complexes that add and remove methyl marks.
Remodeler mechanism and regulation. Jacqueline Wittmeyer has developed a chromatin remodeling system to monitor nucleosome movement and factor binding (Meth. Enz, 2004). Anjanabha Saha has shown that the central function of remodelers is to translocate DNA directionally on nucleosomes (Genes Dev.,. 2002; Nature SMB, 2005) is now testing whether DNA 'waves' travel along the surface of nucleosomes. Collaborations with the Bustamante lab have led to a single-molecule understanding of DNA translocation (in review). Remodelers also bear proteins related to actin, and Heather Szerlong and Kaede Hinata have shown that these proteins form dimeric modules (EMBO, 2003) that regulate remodeling activity (in preparation).
Remodeler targeting and the histone code. Jacqui Wittmeyer is currently testing how histone modifications and acetylation-binding bromodomains affect targeting and/or remodeling by RSC in vitro. Maggie Kasten is investigating how the tandem double bromodomain in the Rsc4 protein reads and coordinates the histone code in vivo and in vitro (EMBO, 2004). A collaboration with Chris Hill’s lab has yielded a high-resolution crystal structure of the Rsc4 tandem bromodomain bound to substrate, and its unique features are the focus of her studies (in preparation). We have shown that RSC occupies hundreds of genes, and redistributes when cellular conditions are altered (Molecular Cell, 2002). Boris Wilson and Tim Parnell are working on how RSC and chromatin interface with signal transduction pathways that sense the environment (Genetics, 2005; and unpublished).
Histone methylation. Alisha Schichter has provided insight on how the major histone methyltransferase is regulated to mark the 5’ ends of active genes with H3K4 trimethylation, showing that the enzyme is autoinhibited by a central domain and activated by an amino-terminal RRM domain (EMBO, 2005). Mat Gordon, Derick Holt, and Anil Panagrahi have purified from S. pombe the complex that removes histone methyl marks, and have uncovered the locations in the genome where this marking/unmarking interplay helps regulate transcription (by genome-wide localization), which has revealed new roles for this complex (in preparation).
Histone variants, DNA methylation, and cancer connections. Dan Richardson and Haiying Zhang use yeast as a model organism for understanding how fusion proteins in humans cause cancer by improperly depositing histone variants and altering acetylation patterns on genes (MCB, 2004). Haiying Zhang has recently used genome-wide localization experiments to reveal the transcriptional strategy of the major histone variant in yeast cells, Htz1 (in press). Kunal Rai is investigating how DNA methylation and chromatin helps regulate development in zebrafish, and has the surprising result that particular DNA methyltransferases are responsible for the development of particular organs. Kimble Frazer works on understanding how chromatin regulates homologous recombination and its relationship to RNA interference in S. pombe.
RNA Polymerase III. We recently utilized genome-wide approaches to define the RNA Polymerase III transcriptome as well as the basic activity-occupancy relationships of this machinery. We are now defining the role of the master regulator of Pol III transcription, Maf1, by genomic and biochemical methods.
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